Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, not only acts as an extra-cellular ligand for the G-protein coupled SIP 1-5 receptors (formerly known as endothelial differentiation gene receptors), but also functions as an intra-cellular second messenger involved in Ca2+ mobilization, proliferation and suppression of apoptosis. Earlier, we and others have demonstrated that platelet derived and exogenously added S1P enhance endothelial barrier function and chemotaxis. Our preliminary experiments suggest that human lung endothelial cells (ECs) rapidly convert exogenous and plasma-derived S1P to intra-cellular S1P; however, it is unclear the molecular mechanisms of intra-cellular S1P signaling that regulate key EC functions. Recently, several lipid phosphate phosphatases (LPPs) and two sphingosine kinases (Sphks) have been cloned and characterized in mammalian cells which may regulate the dynamic balance between extra- and intra-cellular S1P levels. This proposal will address the role of LPPs and Sphks in regulating endothelial calcium homeostasis, proliferation and cytoskeletal reorganization. It is postulated that "LPPs and Sphks regulate intra-cellular generation and signaling of S1P in human lung ECs. Specific Aim 1 will characterize the LPPs and their role in regulating intra-cellular S1P production in the endothelium. Specific Aim 2 will determine the role and regulation of Sphks 1 and 2 in catalyzing the phosphorylation of sphingosine derived by the action of the LPPs on exogenous S1P. Specific Aim 3 will address the cellular targets and functions of intra-cellular S1P. Taken together, these experiments will uncover novel mechanisms by which LPPs and Sphks regulate production of intra-cellular S1P from circulating plasma and also identify intra-cellular targets of S1P involved in calcium homeostasis, secretion, and expression of adhesion molecules, chemotaxis, and wound healing in lung endothelial cells. A better understanding of molecular mechanisms of regulation of LPPs and Sphks in mediating intra-cellular formation of S1P from circulation will provide new insights into possible physiological role of S1P as an intra-cellular second messenger in lung endothelial cells under normal and pathological conditions such as atherosclerosis and asthma.